Conventional chemical pesticides used to control insect pest populations in target crops generally affect beneficial as well as non-beneficial species. Unfortunately through repeated treatments target insect pests have been found to acquire resistance to such chemicals allowing new pest populations to rapidly develop which are resistant to these pesticides. Consequently, new, and potentially more toxic, chemicals must be developed. Furthermore, chemical pesticides pose potential environmental hazards and health concerns, and it is the combination of such factors which has generated considerable concern about the widespread use of broad-spectrum chemical insecticides. This concern has led to research into alternative methods of controlling insect pests.
Biological control represents an important alternative to traditional chemical insect pest control strategies. The two primary approaches of biological control include deployment of naturally occurring organisms which are pathogenic, and, the development of insect resistant crops. With respect to the use of naturally occurring organisms, insect pathogens such as baculoviruses have attracted particular attention because many naturally occurring baculoviruses presently infect insects which are pests of commercially important agricultural and forestry crops. Furthermore, (1) one baculoviruses as a group found solely in arthropods, and (2) individual baculovirus strains are usually restricted in their replication to one or a few species of insects. Consequently, they can be targeted to those species which are sought to be controlled. As such they pose little risk, if any, to man or the environment and can be used without detriment to beneficial insect species. Baculoviruses are therefore potentially valuable as biological control agents.
Baculoviruses are typically packaged in two forms: nucleocapsids may be occluded in the nucleus of infected cells in particles known as "polyhedral inclusion bodies" (PIBs) of which the predominant structural protein is polyhedrin, or, they may bud through the membrane of the infected cell, thereby acquiring a membrane envelope to form non-occluded virus (NOV) particles. Baculovirus sub-groups include nuclear polyhedrosis viruses (NPV), granulosis viruses (GV) and non-occluded baculoviruses. In occluded forms of baculoviruses (NPV and GV sub-groups), the virions' enveloped nucleocapsids are embedded in a crystalline protein matrix. This structure, referred to as an inclusion or occlusion body is the form found extraorganismally in nature and is responsible for spreading the infection between organisms. A characteristic feature of viruses of the sub-group NPV is that many virions are embedded in each occlusion body. The crystalline protein matrix of the occlusion bodies is primarily composed of a single polypeptide which is known as polyhedrin or granulin.
The polyhedrin protein is the product of a viral gene which is expressed very late in the viral reproductive cycle, i.e., well after completion of viral DNA replication. This gene is under the control of a powerful promoter and consequently large quantities of the polyhedrin protein are produced during late infection stages to produce PIBS. Upon cell lysis and insect death PIBs are released into the environment and can last for extended periods until ingested by other insects which will start a new round of viral replication. Non-occluded virus are only viable within the original insect host.
One of the major drawbacks to the use of baculoviruses as a means to achieve insect pest control is the length of time which elapses between the time the baculovirus is ingested by the insect and the time the insect finally dies. Insect larvae infected with such viruses continue feeding for several days after infection and die only when maximum crop damage has occurred.
There have been a number of attempts to provide viruses with the capacity to generate more rapid lethality in the host by introducing genes for neuropeptides or neurotoxins into viral DNA. However, to date, this approach has not provided a solution to the problem, possibly due to difficulty in the expression and processing of products coded by these genes. On the other hand, some success has been generated through introduction into the viral genome of toxin-producing genes from other arthropods, eg., a mite which produces a paralysing toxin when feeding on its host (Tomalski, M. D. & Miller, K., Nature 352, 82-85, 1991; Tomalski and Miller, U.S. Pat. No. 5,266,317; Stewart, L. et al. Nature 352, 85-88, 1991). Unfortunately however, as with the traditional approach using chemical insecticides, there is the possibility of host resistance being developed to the toxin introduced by this methodology. For example, in the case of B. thuringiensis toxin there has been the development of a resistance in the range of target insect pests (see McGaughey, W. H., Agriculture Ecosystems & Environment, 49: 95-102, 1994).
The present invention provides a unique strategy to achieve insect pest control, involving the elimination of the function of a gene essential for insect development in the target insect pest, the effect of which is to almost immediately cease insect growth and development. This is accomplished by introducing a portion of a selected gene, downstream from a powerful viral promoter in an orientation such that when the gene portion is transcribed, an antisense RNA transcript is produced. The complete gene is not required and may not even be fully characterized; a small portion of the antisense transcript is sufficient to completely block translation of the corresponding cellular gene from the host insect. The selected gene is one which encodes a protein which is essential for the growth and development of an insect or other organism from which the gene is obtained.
Antisense strategies have previously been utilized to inhibit expression of a variety of genes responsible for specific human and animal disease. A compilation of these has appeared in a recent issue of The Annals of New York Academy of Sciences (Volume 660, Oct. 28, 1992). Most of these strategies involved the use of antisense oligo-nucleotides targeted to the translation start site of the messenger RNA so as to inhibit ribosomal binding at the start of protein translation, thus cancelling out expression from the gene. Although such strategies have worked, they rely on vast quantities of oligonucleotides to overcome the low efficiency of their entry into living cells, and the effect is soon lost as the oligonucleotides are degraded. In contrast the approach described here involves the use of a carrier virus which not only has privileged access into insect tissues, but is also capable of propagating itself within the host, thus maximizing the effect of the antisense sequences transcribed from its genome.
In the present invention the selected inserted sequence is a gene fragment downstream from the translation start site comprising three conserved stretches of nucleotides, which is inserted into the start of the polyhedrin gene in an orientation such that when transcribed, an antisense RNA transcript is produced. It has been confirmed that abundant transcripts containing the inserted sequence are produced from the polyhedrin promoter and that translation of the polyhedrin protein is blocked as a consequence of the insertion. It is believed that these antisense transcripts block translation of a corresponding native mRNA to produce a protein.
It should be understood that any portion of any gene which will interfere with the production of any cell product which is required for development in the organism should be sufficient for the purposes of this invention. It should also be understood that the gene, or gene portion, can be inserted anywhere downstream from a strong viral promoter so long as it is inserted in an orientation wherein an antisense transcript is produced and in a position to result in the effect of stopping insect growth and development.
The present invention includes all target insect pests which are susceptible to infection by baculoviruses as well as any other insect and vector combination wherein the vector is capable of stopping insect growth and development through implementation of the antisense strategy employed herein. It also includes all genes in a given target insect pest which are required for insect development in the target insect pest.
The present specification includes a method for construction and testing of recombinant baculovirus which produces antisense transcript corresponding to any gene which is required for insect development in the target organism, in an amount sufficient to completely block expression of the corresponding cellular gene from the host insect.